Not applicable.
The invention relates, in a general sense, to assessing the susceptibility of mammals to carcinogenesis.
An individual""s susceptibility to cancer is governed by the individual""s genome and carcinogenic stimuli which the individual encounters in the environment. Although the carcinogenic potentials of many compounds and other stimuli (e.g., ionizing radiation) have been determined, assessing the importance of limiting exposure to such compounds is complicated by the fact that not all individuals are equally susceptible to the carcinogenic effects of the compounds. Thus, the genetic component of carcinogenic susceptibility limits accurate prediction of cancer rates among individuals, even in defined environments.
Prior art observations suggest that up-regulation of the mammalian gene (odc) encoding ornithine decarboxylase is associated with enhanced susceptibility to carcinogenesis (Luk et al., 1984, N. Engl. J. Med. 311:80-83). Other prior art references indicate that overexpression of the odc gene increases susceptibility of mammalian cells for the tumor promotion stage of carcinogenesis, but that over-expression of odc is not, by itself, a sufficient condition for carcinogenesis (Clifford et al., 1995, Cancer Res. 55:1680-1686; Auvinen et al., 1992, Nature 360:355-358; Moshier et al., 1993, Cancer Res. 53:2618-2622; Hibshoosh et al., 1991, Oncogene 6:739-743; O""Brien et al, 1997, Cancer Res. 57:2630-2637).
It is known that there are at least two alleles of the human odc gene, which have been observed as different PstI restriction fragment length polymorphisms (RFLP; Hickok et al., 1987, DNA 6:179-187).
It is believed that the odc gene product, ornithine decarboxylase, is involved in establishing cellular polyamine levels, and that the susceptibility of a tissue to carcinogenesis is related to polyamine levels in the cells of the tissue. Transcription of the odc gene is activated by the protein designated Myc (Bello-Fernandez et al., 1993, Proc. Natl. Acad. Sci. USA 90:7804-7808; Tobias et al., 1995, Oncogene 11:1721-1727; Wagner et al., 1993, Cell Growth Diff. 4:789-883). Such regulation is effected by binding of Myc/Max heterodimers to one or more of the three Myc binding elements (also called xe2x80x9cE-boxesxe2x80x9d) in the human odc gene. These E-boxes are located at about nucleotide residues xe2x88x92489 to xe2x88x92484 (relative to the transcription start site as defined by Moshier et al., 1992, Nucleic Acids Res. 20:2581-2590) in the odc 5xe2x80x2-promoter region and at nucleotide residues +288 to +293 and +322 to +327 in the first intron of the odc gene. The fact that the E-box located in the promoter region of the human gene is not conserved in the rat and mouse genes, suggests that the other two E-boxes are the physiologically important regulatory elements.
In normal cells, expression of c-myc (the gene encoding Myc protein) is associated with cell cycle progression. Lack of c-myc expression permits cells to withdraw from the cell cycle and cease differentiating. Furthermore, constitutive c-myc expression promotes continuous cell cycle progression independent of growth factors (Rapp et al., 1985, Nature 317:434-438). Over-expression of c-myc is a common characteristic of many (if not most) human tumors. Prior art studies have correlated c-myc expression and odc expression in human tumors, but not in surrounding normal (i.e., non-tumor) tissue (Gan et al., 1993, J. Histochem. Cytochem. 41:1185-1196; Mori et al., 1996, Cancer 77{8 Suppl.}:1634:1638; Mimori et al., 1997, Dis. Colon Rectum 40:1095-1100). These studies suggest that deregulation of c-myc expression may lead to deregulation of odc expression.
Numerous stimuli are known which induce deregulation of c-myc expression (Schwab et al., 1984, Proc. Natl. Acad. Sci. USA 81:4940-4944; Payne et al., 1982, Nature 295:209-214; Klein et al., 1985 Immunol. Today 6:208-215). Because deregulated Myc transactivates odc expression, these stimuli can be predicted to induce deregulation of odc expression in general. However, until the present invention, it has not been known whether either of the alleles of odc is more susceptible to transactivation by Myc, and thus the genetic component of carcinogenic susceptibility of an individual could not be ascertained.
The invention relates to a method of assessing the relative susceptibility of a mammal (e.g., a human) to an epithelial cancer. The epithelial cancer can, for example be a skin cancer (e.g., a squamous cell carcinoma), a cancer of the digestive system, an esophageal cancer, a gastric cancers, a colon cancer, a prostate cancer, a breast cancer, an hematopoietic cancer, a lung cancer, a melanoma, or a cervical cancer. The method comprises determining whether the mammal comprises an A-allele of the odc gene. If the mammal comprises the A-allele, then the mammal has a greater susceptibility to the epithelial cancer than a mammal of the same type which does not comprise the A-allele.
In one embodiment, determining whether the mammal comprises an A-allele of the odc gene comprises amplifying a reference portion of the mammal""s genome and identifying the reference portion as corresponding to either the A-allele or the G-allele. The reference portion comprises a region of the odc gene comprising at least one nucleotide residue that is polymorphic with respect to the A- and G-alleles. By way of example, the reference portion can be amplified using a pair of primers having nucleotide sequences SEQ ID NOs: 3 and 4. Other primer pairs that can be used to amplify the reference portion include a primer pair having the nucleotide sequences SEQ ID NOs: 16 and 17, and a primer pair having the nucleotide sequences SEQ ID NOs: 18 and 19. Optionally, the reference portion can be further amplified using a pair of nested primers (e.g., primers having nucleotide sequences SEQ ID NOs: 5 and 6 if primers having SEQ ID NOs: 3 and 4 are used for the initial amplification). By way of example, the reference portion can comprise a region (e.g., intron 1) of the odc gene, such as nucleotide residues +278 to +336 relative to the transcription start site. The reference portion can, for example, have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 24, and 25). The region preferably comprises the nucleotide residue located at a position selected from the group consisting of xe2x88x923175, xe2x88x923004, xe2x88x921936, +263, +317, +5294, +5915, +6697, +7487, and +7886 relative to the transcription start site, of the odc gene.
An oligonucleotide probe can be annealed with a target portion of the mammal""s genome prior to amplifying the reference portion. Preferably, the target portion includes a target nucleotide residue that is polymorphic with respect to the A- and G-alleles of the odc gene. By way of example, the target portion can include the target nucleotide residue located at a position selected from the group consisting of xe2x88x923175, xe2x88x923004, xe2x88x921936, +263, +317, +5294, +5915, +6697, +7487, and +7886, relative to the transcription start site, of the odc gene. In one embodiment, the probe is attached to a surface. The probe can, optionally, comprise a fluorescent label such as FAM, TET, rhodamine, VIC, JOE, or Hex. The probe can further comprise a fluorescence quencher such as TAMRA or DABCYL. For example, the probe can have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 20, and 22, and can comprise both a fluorescent label and a fluorescent quencher. When the probe comprises both the label and the quencher, one is preferably attached to the probe within 10 nucleotide residues of the 3xe2x80x2-end of the probe and the other is preferably attached to the probe within 10 nucleotide residues of the 5xe2x80x2-end of the probe. The reference portion can be amplified using a DNA polymerase having 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity, such as Thermus aquaticus DNA polymerase. Preferably, the probe has a length from 15 to about 30 nucleotide residues and the size of the reference portion is not more than about 100 nucleotide residues.
In another embodiment of this method of the invention, determining whether the mammal comprises an A-allele of the odc gene comprises contacting a polynucleotide derived from the mammal""s genome with a molecular beacon probe. The probe has a targeting portion which is complementary to a target region of the odc gene, and the target region includes a nucleotide residue that is polymorphic with respect to the A- and G-alleles. For example, the target region can include the nucleotide residue located at a position selected from the group consisting of xe2x88x923175, xe2x88x923004,xe2x88x921936, +263, +317, +5294, +5915, +6697, +7487, and +7886, relative to the transcription start site, of the odc gene. Preferably, the targeting portion is completely complementary to the target region of the A-allele of the odc gene and has a length from about 20 to about 40 nucleotide residues. For example, the targeting portion of the first probe can have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 20, and 22, and the targeting portion of the second probe can have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 21, and 23. The target region can, for example, comprise about 20 to about 30 consecutive nucleotide residues of a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 24 and 25. The polynucleotide can, for example, be selected from the group consisting of a chromosome of the mammal, a chromosomal fragment of the mammal, and an amplified portion of the genome of the mammal. Optionally, the method of this embodiment can further comprise contacting the polynucleotide with a second molecular beacon probe. The second probe has a targeting portion which is completely complementary to a target region of the G-allele of the odc gene. One or both of the probes can be attached to a discrete region of a surface or the first probe can be attached to one surface or particle and the second probe can be attached to a different surface or particle.
The invention also relates to an isolated polynucleotide having a length of at least about 15 nucleotide residues and being either homologous with or complementary to a portion of a mammalian (e.g., human) odc gene. The portion of the odc gene includes a nucleotide residue that is polymorphic with respect to the A- and G-alleles. By way of example, the nucleotide residue can be located at a position selected from the group consisting of xe2x88x923175, xe2x88x923004, xe2x88x921936, +263, +317, +5294, +5915, +6697, +7487, and +7886, relative to the transcription start site, of the odc gene. In one embodiment, the nucleotide residue at position +317 is a guanine residue, the nucleotide residue at position xe2x88x923175 is a thymine residue, and the nucleotide residue at position xe2x88x923004 is a cytosine residue. By way of example, the isolated polynucleotide can have the nucleotide sequence of one of SEQ ID NOs: 14, 15, and 20-23.
The invention further relates to a kit for assessing susceptibility of a mammal to an epithelial cancer. The kit comprises a first oligonucleotide probe which anneals specifically with a target portion of the mammal""s genome and a first primer for amplifying a reference portion of the odc gene. The target portion includes a target nucleotide residue that is polymorphic with respect to the A- and G-alleles. For example, the target nucleotide residue can be the nucleotide residue located at a position selected from the group consisting of xe2x88x923175, xe2x88x923004, xe2x88x921936, +263, +317, +5294, +5915, +6697, +7487, and +7886, relative to the transcription start site, of the odc gene. The first probe comprises a fluorescent label and a fluorescence quencher attached to separate nucleotide residues thereof. The reference portion also includes the target nucleotide residue. The kit can further comprise a DNA polymerase having 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity. In one embodiment, the first probe is completely complementary to the target portion if the nucleotide residue located at position +317 is A, and the kit further comprises a second oligonucleotide probe which is completely complementary to the target portion if the nucleotide residue located at position +317 is G and a second primer for performing PCR amplification of at least the target portion in conjunction with the first primer. For example, the kit can comprise i) first and second oligonucleotide probes having nucleotide sequences SEQ ID NOs: 14 and 15, 20 and 21, or 22 and 23; ii) first and second amplification primers having nucleotide sequences SEQ ID NOs: 12 and 13, 16 and 17, or 18 and 19; and iii) Taq DNA polymerase. In one embodiment, the first and second probes are attached to discrete regions of a surface or to a plurality of surfaces such that the first probe is attached to a surface or particle discrete from the second probe. In another embodiment, the first probe is completely complimentary to the target portion if the nucleotide residue located at position xe2x88x923175 is C. This kit can further comprise a second oligonucleotide probe which is completely complementary to the target portion if the nucleotide residue located at position xe2x88x923175 is T or a second primer for performing PCR amplification of at least the target portion when used with the first primer. In yet another embodiment, the first probe is completely complimentary to the target portion if the nucleotide residue located at position xe2x88x923004 is G. This kit can further comprise a second oligonucleotide probe which is completely complementary to the target portion if the nucleotide residue located at position xe2x88x923004 is C or a second primer for performing PCR amplification of at least the target portion when used with the first primer.
In another aspect, the invention relates to a method of assessing whether a test compound is an inhibitor of carcinogenesis. This method comprises assessing ornithine decarboxylase activity in a cell comprising an A-allele of the human odc gene in the presence of an inducer of carcinogenesis and in the presence or absence of the test compound. If ornithine decarboxylase activity is lower in the presence of the test compound than in the absence of the test compound, then the test compound is an inhibitor of carcinogenesis.
The invention also relates to a method of assessing whether a test compound is an inducer of carcinogenesis. This method comprises assessing ornithine decarboxylase activity in a cell comprising an A-allele of the human odc gene in the presence of an inducer of carcinogenesis and in the presence or absence of the test compound. If ornithine decarboxylase activity is greater in the presence of the test compound than in the absence of the test compound, then the test compound is an inducer of carcinogenesis.